INTRODUCTION:

The Asteraceae family includes the plant known as sweet leaf, Stevia rebaudiana Bertoni. This perennial herb, native to Brazil and Paraguay, thrives in humid environments¹^,². Stevia leaves contain sweet steviol glycoside components, with stevioside accounting for 4–13 percent of the total.There are additional steviol glycosides as well, including dulcoside A (0.4-0.7%), rebaudioside A (2-4%), and rebaudioside C (1-2%). The age, growth location, and time of harvest all affect the amount of each steviol glycoside that distinguishes stevia from other plants. Stevia is a semi-humid subtropical plant that grows in regions with -6 to 43.8ºC temperatures and 1500 to 1800mm of annual precipitation³. 4mg/kg of steviol glucosides was determined to be the tolerable daily consumption by the Joint FAO/WHO Committee on Food Additives (JECFA)⁴.

Apart from glycosides, phenolic acids, flavonoids, fatty acids, proteins, and vitamins are also present in stevia leaves. Stevia extracts have exhibited antibacterial, anti-hypertensive, anti-tumor, anti-inflammatory, liver-protective, Anti hyperlipidemic impact, immuneboosting, and antioxidant characteristics⁵^,⁶, reduce hyperglycemia Stevia's high concentration is thought to be responsible for its antioxidant properties. Flavonoids and phenolic acids⁷^,⁸^,⁹. This study uses the following solvents in different solutions (deionized distilled water, 95% ethanol, and a water-propylene glycol mixture in a 4:1 ratio) to prepare extracts of Stevia leaves, which are then subjected to microscopic observation, phytochemical screening, and analysis of the total content of phenols and flavonoids and antioxidant activity. These extracts have potential applications in cosmetics and nutritional supplements.

MATERIALS AND METHODS:

Materials:

Chemicals and Reagents:

All chemicals and reagents in the study were of analytical grade. Methanol (Biosolve Chimie SARL, France), ethanol and aluminum chloride (Honeywell, Germany),ethyl acetate (Surechem Products Ltd, UK), folin-Ciocalteu Detector (Merck/Germany), gallic acid, ascorbic acid, quercetin (Prolabo), DPPH (diphenylpicrylhydrazyl) reagent (TCI, Japan), Anhydrous sodium carbonate (Sham Lab, Syria), petroleum ether (Panreca Company), aluminum chloride (Riedel de Haen), hydrochloric acid (Sham), magnesium metal (Chem lap), lead acetate (Sham), iron chloride (Riedel de Haen), chloroform (Fisher), ammonia (Sham), vanillin (Eternal Pearl brand), boric acid (Panreac).

Instruments:

Light microscope (OLYMPUS OPTICAL, Model CHT, Japan), Sensitive electronic balance (Shimadzu AX200, Japan), UV-VIS Spectrophotometer ((T80+), PG Instruments, United Kingdom), rotatory evaporator (RV 10 digital IKA, Germany).

Methods:

Preliminary Phytochemical Screening:

Preliminary phytochemical investigation of powder of leaves of Stevia rebaudiana was done by using several reagents assigned for the detection saponins, flavonoids, coumarins, anthraquinones, tannins, alkaloids, and cardiac glycosides on dry plant leaves.¹⁰^,¹¹^,¹²^,¹³

Sourcing and preparation of extracts:

Dry stevia leaves were purchased from Belqis Nurseries in Damascus, Syria. Leaves were harvested in August and dried in the shade outdoors. Three extracts were prepared from dry stevia leaves. Aqueous extract - 95% ethanolic extract - extract from a mixture of propylene glycol with water in a ratio of 4:1. Ethanol and water are the optimal solvents for extracting polyphenols. 5g of dry leaves were crushed with 20ml of solution for 5 minutes in a mortar. Subsequently, the mortar content was transferred to a beaker with 80ml of solution and placed on a magnetic stirrer for 3hours. The propylene glycol extract was filtered with water and stored in an opaque container at a temperature of 4°C. The aqueous and ethanolic extracts were dried after filtration using a rotary evaporator and stored at a temperature of 4°C.

Microscopic Characteristics:

The powder was treat with 20% chloral hydrate in glycerin with heating then studied using the light microscope (OLYMPUS OPTICAL, Model CHT, Japan) on optical magnification X10 and X40.

Determination of the total phenolic content in plant extracts:

Slight adjustments were made to the procedure described by Ahmed et al. to ascertain the total phenolic contents of the samples.In summary, 20 microliters of the sample (gallic acid or extract), 1.58 millilitres of distilled water, and 100 microliters of 10% FCR1 were combined. A 20% Na2CO3 solution containing 300μl was added after 5 minutes. The reaction tubes were covered, vortexed, and left at room temperature for 45 minutes in the dark. Using a UV-visible spectrophotometer (Model: T80+, PG Instrument Ltd, United Kingdom) set to the maximum absorbance wavelength of 765nm, the absorbance reading of each sample was determined¹⁴^,¹⁵^,¹⁶. It took two millilitres of distilled water to produce the blank. The equation of the linear calibration curve for gallic acid (y= 0.0028x + 0.0285) (R2 = 0.9964) was used to determine the total phenolic content. Gallic Acid Equivalents (GAE) per gramme of dried leaves (DL) were used to express all results. Every measurement was made three times¹⁷. standard deviations plus the mean were used to express the results. Gallic acid (5000mg/l) was dissolved in water to create a stock solution, which was then used to create a series of dilutions ranging from 0 to 500mg/l.

Ten milligrammes of each extract were dissolved in five millilitres of distilled water to create the leaf extracts, which were then diluted to five millilitres with one millilitre of propylene glycol extract in water.

Determination of the total flavonoids content in plant extracts:

By dissolving 10mg of each extract in 5ml of methanol, the extracts of Stevia leaves were created at a concentration of 2mg/l. The aluminium chloride colorimetric approach, as reported by Al-Metrin et al.¹⁸, was slightly modified in order to determine the total flavonoid concentration. In summary, the following ingredients were combined: 2.8ml of distilled water, 0.1 ml of 1M sodium acetate, 0.1ml of 10% methanolic aluminium chloride solution, and 2ml of the sample (quercetin or extract). After covering and vortexing the reaction tubes, they were allowed to sit at room temperature in the dark. The aluminum-flavonoid complex that results from the reaction is yellow. A UV-visible spectrophotometer (Model: T80+, PG Instrument Ltd, United Kingdom) was used to assess each sample's absorbance reading after 30minutes at its maximum absorbance wavelength of 415nm¹⁹^,²⁰. Two millilitres of 100% methanol were used to create the blank. Using the calibration curve equation for quercetin²¹.(y= 0.0141x + 0.048) (R2 = 0.9963), the total flavonoid concentration was determined.For each gramme of dried leaves (DL), the results were represented as milligrammes of quercetin equivalents (QE). Every measurement was made three times. The mean±standard deviation was used to express the results.Two millilitres of propylene glycol extract were obtained, and 10 millilitres of aqueous extract and 10 millilitres of ethanolic extract were dissolved in methanol to yield concentrations of one millilitre per millilitre (1%). Every extract was made in three replicates, and each extract's absorption was assessed three times.

In vitro antioxidant activity assessment of Stevia extracts:

Diphenylpicrylhydrazyl, or DPPH, is a stable free radical that can be used to measure the antioxidant potential of stevia extracts at room temperature. This technique evaluates the free radicals capacity for scavenging. It is based on electron transport, which gives ethanol a violet solution. The presence of antioxidant molecules neutralises this free radical by giving it hydrogen, which stabilises the free radical. The solution's implied change in colour from violet to yellow serves as an indicator of this. Using a spectrophotometer to measure the drop in colour intensity, free radicals capacity for scavenging was evaluated²²^,²³. For each of the three extracts, a standard series of ascorbic acid (positive control) was generated along with a range of varied concentrations.Then, 200μl of the extract solution or ascorbic acid was added to a test tube, and 2ml of DPPH solution was added after that,Next, a spectrophotometer was used to measure the absorbance of the tubes at a wavelength of 517nm⁹^,²⁴.

The percentage of free radical inhibition was calculated using the following formula:

IDPPH% = [(Ab - Aa) / Ab] x 100

Aa Absorption of DPPH solution with extract/Ab absorbance of only DPPH solution without extract

A fresh solution of DPPH was prepared by dissolving 2.25mg in 50ml of absolute methanol to obtain a concentration of 45μg/ml and kept in a dark place to prevent its deterioration by light. Preparation of the mother solution of ascorbic acid in concentration of 0.7 mg/ml. preparation of the mother solution of aqueous and ethanolic extracts in concentration of 5mg/mL of each, the propylene glycol extract in water was prepared by diluting 1mL in 9mL methanol to achieve a concentration of 5mg/ml. the Preparation of the standard series of ascorbic acid: The following concentrations were prepared from the stock solution: 0.012-0.023-0.047-0.064-0.07mg/ml. Aqueous extract concentrations: (0.25-0.5-1-2-3-4)mg/ml. Ethanol extract concentrations: (0.25-0.4-1.5-2)mg/ml. Concentrations of propylene glycol extract in water: (0.25-1-2-3)mg/ml

RESULT:

Results of preliminary phytochemical screening:

The results of the preliminary investigation for secondary metabolites are shown in Table 1. The (+) mark indicates a positive test result, while the (−) indicates a negative result. Leaves of Stevia rebaudiana contain phenols, flavonoids and coumarins, while saponins, anthraquinones, tannins, alkaloids, and cardiac glycosides are absent.

Table 1: the results of preliminary investigation tests for secondary metabolites

Secondary metabolites	Test name	Result
Saponins	Foam test	-
Saponins	Reaction with aromatic aldehydes	-
Flavonoids	Reaction with aluminum chloride	+
	Shinoda	+
	Wilson-Taubouk	+
Coumarins	Fluorescence	+
Anthraquinons	Borntrager	-
Tanins	FeCl3	+
	Reaction with Lead acetate	-
	Gelatin precipitation	-
Alkaloids	Dragendroff	-
	Mayer	-
	Wagner	-
	Hager	-
Cardiac glycosides	Keller-kiliani	-
	kedde	-
	Baljet	-

Microscopic study results:

When studying the leaves and flowers microscopically, the following distinctive elements were noted:

1 Leaves:

a. Epidermis with pores of an irregular pattern

b. distinctive multicellular tentacle operculum.

2. Flowers:

c. Pollen grains are small, measuring 22.5

d. par tentacle (Figure.1).

Figure. 1: Microscopiacal charcterstics of Stevia rebaudiana a. Epidermis with pores of an irregular pattern of leaves, b. distinctive multicellular tentacle operculum of leaves, c. Pollen grains are small measuring 22.5 µm d. par tentacle.

Determination of the total phenolic content in plant extracts:

Results of the titration of total phenols in the three extracts:

Aqueous extract: 4.55±0.857mg/g dry plant.

Ethanolic extract: 8.867±0.5046mg/g dry plant.

Propylene glycol extract with water: 2.354±0.762mg/g dry plant.

The standard curve of gallic acid standard series linking concentration and absorption, and the linear relationship between them can be seen in (Figure.2).

Figure. 2: Standard calibration curve for total phenolic content for standard gallic acid

The total phenolic content in the ethanolic extract is the highest among the three extracts (Figure.3).

Figure. 3: Quantitative estimation of total phenolic content (TPC)

Determination of the total flavonoids content in plant extracts:

Aqueous extract: 2.89±0.2164mg/g dry plant.

Ethanolic extract: 2.523±0.013mg/g dry plant.

Propylene glycol extract with water: 0.156±0.000115 mg/g dry plant.

The standard curve of Quercetin standard series linking concentration and absorption, and the linear relationship between them can be seen in (Figure.4).

Figure. 4: Standard calibration curve for total flavanoids content for standard quercetin

The content of total flavonoids in the aqueous extract is the highest among the three extracts (Figure.5).

Figure. 5: Quantitative estimation of total flavonoid.

Determine the scavenging ability of free radicals:

The absorption of the standard series tubes for ascorbic acid and the absorption of the negative control tube were measured at a wavelength of 517nm. Subsequently, the percentage of free radical scavenger activity was calculated. The standard series graph illustrating the relationship between the concentration and effectiveness of ascorbic acid was plotted, showing a linear correlation between them. The IC₅₀ for the acid was then calculated. Ascorbic acid is determined by the straight-line equation (representing the concentration needed to scavenge 50% of free radicals).

The IC₅₀ for ascorbic acid was 0.041mg/ml.

The previous steps were repeated for the three extracts. A graphic curve was drawn for each extract, and the IC₅₀ values were calculated based on the graphic curve equation.

Figure. 6: scavenging DPPH• by ascorbic acid

Free radical scavenging activity of the aqueous extract:

IC₅₀ = 1.39mg/mL

Figure. 7: scavenging DPPH• by aqueous extract

Free radical scavenging of the of ethanolic extract:

IC₅₀ = 0.54mg/mL

Figure. 8: scavenging DPPH• by ethanolic extract

Free scavenging scavenger activity of PG with water:

IC₅₀ = 1.537mg/mL

Figure. 9: scavenging DPPH• by PG+water extract

DISCUSSION:

The results of our study are consistent with the results of most microscopic studies of the presence of Distinctive multicellular tentacle operculum in the leaves, and are consistent with the results of a study conducted in Iraq regarding the Epidermis with pores of an irregular pattern²⁵. And and are consistent with the results of a study conducted in Chile regarding the Upar tentacle²⁶. This study determined for the first time the pollen grains measurement as 22.5μm.

The results of our study are consistent with the findings of the majority of previous studies regarding the presence of flavonoids and coumarins in plant leaves.

The results of the current study differed from those of some previous studies regarding the plant's secondary metabolite content. This study yielded positive results for the presence of tannins, saponins, and anthraquinones in stevia leaves, which contrasts with findings from studies conducted in India²⁷. Comparison of the free radical scavenging activity of the three extracts: We observed that the free radical scavenging ability increases with the concentration of the extract, as indicated by the IC₅₀ values of the three extracts. We also observed that the ethanolic extract exhibited the highest capacity to scavenge free radicals, followed by the aqueous extract, and then the propylene glycol extract with water. This trend is directly related to the phenol content of the extracts, as the highest concentration was found in the ethanolic extract, followed by the aqueous extract, and then the PG extract with water.

We conclude from the previous results that the plant has an average ability to scavenge free radicals compared to ascorbic acid, which is one of the most powerful antioxidants. This effectiveness is due to the stevia plant's content of multiple phenolic and flavonoid compounds. The results of the current study were consistent with those of a study conducted on stevia in Bangladesh. In the Bangladeshi study, the total phenol content of the ethanolic extract exceeded that of the aqueous extract, and the ethanolic extract demonstrated a higher capacity to scavenge free radicals¹.

The results of our study were also consistent with a study conducted in India. Dry stevia leaves had the highest percentage of phenolic content in the ethanolic extract, but it did not correspond to the antioxidant capacity. The aqueous extract exhibited a greater antioxidant capacity compared to the ethanolic extract²⁸.

The results of our study were also consistent with a study conducted in India. Dry stevia leaves had the highest percentage of phenolic content in the ethanolic extract, but this did not correspond to the antioxidant capacity. The aqueous extract exhibited a higher antioxidant capacity compared to the ethanolic extract²⁸

The results of our study did not agree with those of a study conducted in Poland. In the Polish study, the propylene glycol extract with water exhibited the highest content of phenols, flavonoids, and antioxidant capacity, followed by the aqueous extract with ethanol⁷.

This variation may be attributed to differences in experimental conditions, sources of the stores, plant origins, cultivation conditions, climatic factors, and soil types.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

REFERENCES:

1. Sultana C, Azad MA, Rahman MM, Muhit MA, Rahman SA. Phytochemical and Biological Investigation of Stevia rebaudiana (Bert.) Leaves Grown in Bangladesh. Dhaka Univ J Pharm Sci. 2020;19(2):191-197. doi:10.3329/dujps.v19i2.50636

2. Pillwan SN, Thool ND, Chopkar SH, Mathankar SP, Pise SA, Pise AG. to Study Extraction, Phytochemical Screening and Formulation from Stevia rebaudiana bertoni. Res J Pharmacogn Phytochem.

3. Junaedi EC, Lestari K, Muchtaridi M. Recent Quantitative Analysis of Stevioside from Stevia Rebaudiana ( Bertoni ): A Review. Res J Pharm Technol. Published online 2020:1-7.

4. Perrier JD, Mihalov JJ, Carlson SJ. FDA regulatory approach to steviol glycosides. Food Chem Toxicol. 2018;122:132-142. doi:10.1016/j.fct.2018.09.062

5. Murtaza M, Tajammal A, Hamza M, Mirza W, Nazir A, Hanif I. A Short Review on Synthetic Methodologies of Flavonoids. asian J Pharm Technol. Published online 2022.

6. Amina B benzid, Roukia H, Amahammed H, Ahlem T, Sabrina B, Chahrazed B. Optimization of Extraction conditions of the Polyphenols , Flavonoids and the Antioxidant activity of the plant Ammosperma cinereum (Brassicaceae) through the Response Surface Methodology (RSM). Asian J Res Chem. Published Online 2020.

7. Gaweł-Bȩben K, Bujak T, Nizioł-Łukaszewska Z, et al. Stevia rebaudiana Bert. leaf extracts as a multifunctional source of natural antioxidants. Molecules. 2015; 20(4): 5468-5486. doi:10.3390/molecules20045468

8. Gupta E, Mohammed A, Purwar S, Rizvi SI, Sundaram S. Diminution of oxidative stress in alloxan-induced diabetic rats by Stevia rebaudiana . Res J Pharmacogn Phytochem. 2017;9(3):158. doi:10.5958/0975-4385.2017.00030.9

9. Valli G, Jeyalakshmi M. Preliminary Phytochemical and Antioxidant Study of Odina woodier Leaf Extract. asian J Pharm Res.:2-5.

10. الشماع عصام, حسن آغا محمد عصام اأس. العقاقير وكيمياء العقاقير(2).

11. Shah and Seth. Textbook of Pharmacognosy and Phytochemistry.; 2010.

12. Rao N, Mittal S, Menghani E. Assessment of Phytochemical Screening, Antioxidant and Antibacterial Potential of the Methanolic Extract of Ricinus communis l. Asian J Pharm Technol.

13. Morris SS, Jeyabalan G, Jha AK, Verma S, Swarnkar Y. Phytochemical Screening and In- vitro Athelmintic activity of Seed Extracts of Plants Carum carvi of Family Apiaceae. Asian J Res Pharm Sci.

14. Jain AP, Choudhary GP. In vitro Antioxidant potential of Sphaeranthus indicus and Abrus precatorius leaves extracts. Res J Pharmacogn Phytochem. 3-8.

15. Tiwari P, Patel RK. Estimation of Total Phenolics and Flavonoids and Antioxidant Potential of Ashwagandharishta Prepared by Traditional and Modern Methods. Asian J Pharm Anal. Published Online 2013:1-6.

16. Allaoui M, Belfar ML. Comparison of Antioxidant activity Total Phenolic , and Total Flavonoids content of Butanolic extracts from Haloxylon scoparium and Traganum nudatum. Asian J Res Chem. 2012; November

17. Ahmed F. Antioxidant activity of Ricinus Communis. Org Med Chem Int J. 2018;5(4). doi:10.19080/omcij.2018.05.555667

18. Al-Mterin M, Aboalhaija N, Abaza I, Kailani M, Zihlif M, Afifi F. Chromatographic Analysis (LC-MS and GC-MS), Antioxidant Activity, Total Phenol and Total Flavonoid Determination of Ononis natrix L. Grown in Jordan. Jordan J Chem. 2021;16(1):31-39. doi:10.47014/16.1.4

19. Kumar MS, Balachandran S. Influence of Incubation Temperatures on Total Phenolic, Flavonoids Content and Free Radical Scavenging Activity of Callus from Heliotropium indicum L . Asian J Pharm Res.

20. Wadkar KA, Magdum CS. Evaluation of Total Phenolic Content , Flavonoid Cont ent and Antioxidant Activity of Stem Bark of Careya arborea Roxb. Res J Pharmacogn Phytochem.

21. Dubey R, Rajhans S, Mankad AU. Preliminary Phytochemical Screening , Quantitative Estimation of Total Phenolic and Flavonoid Content of Jatropha gossypiifolia (L.). Res J Pharmacogn Phytochem. Published online 2019.

22. Garcia EJ, Alencar SM De, Reis A, Loguercio AD, Helena R, Grande M. Antioxidant Activity by DPPH Assay of Potential Solutions to be Applied on Bleached Teeth. 2012; 23:22-27.

23. Jadhav GB, Saudagar RB. Free radical Scavenging and Antioxidant Activity of Punica granatum Linn. Asian J Res Pharm Sci.:1-4.

24. Belfar A, Bensaci C, Belguidoum M. Evaluation of Antioxidant Capacity by Cyclic Voltammetry of Phoenix dactylifera L. (date palm). asian J Res Chem. Published online 2022.

25. Al-taie AT. Study Of Some Anatomical Features Of Stevia Rebaudiana ( Bertoni ) Bertoni Study Of Some Anatomical Features Of Stevia Rebaudiana ( Bertoni ) Bertoni Grown In Iraq. 2022;(December 2021):10-14. doi:10.37652/JUAPS.2021.15

26. Morales V, Moreira-muñoz A. The genus Stevia (Eupatorieae, Asteraceae) in Chile: a Taxonomical and morphological analysis. 2016; 282(1): 1-18.

27. Tadhani M, Subhash R. Preliminary studies on Stevia rebaudiana leaves: proximal composition, mineral analysis and phytochemical screening. J Med Sci. Published online 2006.

28. Sri K V. In Vitro Evaluation of Total Phenol and Antioxidant Activity of Achillea Millefolium and Stevia Rebaudiana.

Received on 07.05.2024 Revised on 09.08.2024

Accepted on 14.10.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):111-116.

DOI: 10.52711/0974-360X.2025.00017

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